Y. Tanaka performed the technical improvement of Y2H. This work is supported by JSPS KAKENHI Grant Number JP22K06336 and the Institute for Fermentation, Osaka.

1. Construction of the mutant library
<ol>
	<li>Set up the polymerase chain reaction (PCR) (an example is shown below). Generally, prepare 50 &#181;L of the reaction, which is divided into ten aliquots of 5 &#181;L, and amplify the target gene fragment in a PCR machine4. 
	NOTE: Users should select an appropriate polymerase to efficiently introduce mutations. If the DNA fragment to be amplified is short, then a polymerase with a higher error rate, such as Taq polymerase, wh...

Biomolecular Interaction Studies Using Yeast Two-Hybrid Technique 

<ol>
	<li>Fields, S., Song, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+genetic+system+to+detect+protein-protein+interactions.">A novel genetic system to detect protein-protein interactions.</a> Nature. 340 (6230), 245-246 (1989).</li><li>Leanna, C. A., Hannink, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reverse+two-hybrid+system:+A+genetic+scheme+for+selection+against+specific+protein/protein+interactions.">The reverse two-hybrid system: A genetic scheme for selection against specific protein/protein interactions.</a> Nucleic Acids Res. 24 (17), 3341-3347 (1996).</li><li>Vidal, M., Brachmann, R. K., Fattaey, A., Harlow, E., Boeke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+two-hybrid+and+one-hybrid+systems+to+detect+dissociation+of+protein-protein+and+DNA-protein+interactions.">Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions.</a> Proc Natl Acad Sci U S A. 93 (19), 10315-10320 (1996).</li><li>Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+the+isolation+of+interaction-null/impaired+mutants+using+the+yeast+two-hybrid+technique.">An efficient method for the isolation of interaction-null/impaired mutants using the yeast two-hybrid technique.</a> Genes Cells. 24 (12), 781-788 (2019).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: A laboratory manual. 4th edn. , (2012).</li><li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat Methods. 6 (5), 343-345 (2009).</li><li>Inoue, H., Nojima, H., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+Escherichia+coli+with+plasmids.">High efficiency transformation of Escherichia coli with plasmids.</a> Gene. 96 (1), 23-28 (1990).</li><li>Birnboim, H. C., Doly, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+alkaline+extraction+procedure+for+screening+recombinant+plasmid+DNA.">A rapid alkaline extraction procedure for screening recombinant plasmid DNA.</a> Nucleic Acids Res. 7 (6), 1513-1523 (1979).</li><li>Bartel, P. L., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein-protein+interactions+using+two-hybrid+system.">Analyzing protein-protein interactions using two-hybrid system.</a> Meth Enzymol. 254, 241-263 (1995).</li><li>Hollenberg, S. M., Sternglanz, R., Cheng, P. F., Weintraub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+new+family+of+tissue-specific+basic+helix-loop-helix+proteins+with+a+two-hybrid+system.">Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system.</a> Mol Cell Biol. 15 (7), 3813-3822 (1995).</li><li>Gietz, R. D., Woods, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+yeast+by+lithium+acetate/single-stranded+carrier+DNA/polyethylene+glycol+method.">Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.</a> Meth Enzymol. 350, 87-96 (2002).</li><li>Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+reliable+protein+extraction+from+yeast.">Rapid and reliable protein extraction from yeast.</a> Yeast. 16 (9), 857-860 (2000).</li><li>Van Deursen, F., Sengupta, S., De Piccoli, G., Sanchez-Diaz, A., Labib, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mcm10+associates+with+the+loaded+DNA+helicase+at+replication+origins+and+defines+a+novel+step+in+its+activation.">Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation.</a> EMBO J. 31 (9), 2195-2206 (2012).</li><li>Watase, G., Takisawa, H., Kanemaki, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mcm10+plays+a+role+in+functioning+of+the+eukaryotic+replicative+DNA+helicase,+cdc45-mcm-gins.">Mcm10 plays a role in functioning of the eukaryotic replicative DNA helicase, cdc45-mcm-gins.</a> Current Biology: CB. 22 (4), 343-349 (2012).</li><li>Miyazawa-Onami, M., Araki, H., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-initiation+complex+assembly+functions+as+a+molecular+switch+that+splits+the+mcm2-7+double+hexamer.">Pre-initiation complex assembly functions as a molecular switch that splits the mcm2-7 double hexamer.</a> EMBO Rep. 18 (10), 1752-1761 (2017).</li></ol>

This article describes how to isolate interaction-null/impaired mutants using the Y2H assay. Such mutants are powerful tools to analyze the function of a protein of interest. To isolate such mutants, rY2H assays were developed previously by modifying the Y2H host strain2,3. However, they have not greatly reduced the amount of labor. By contrast, mutants can be isolated with this method without a significant amount of labor. In this method, modification of the Y2H...

Interactions between biomolecules are the most basic part of biological phenomena. Protein-protein interactions constitute a significant part of such interactions. Therefore, identification of the interaction partner(s) of a protein of interest is critical to further elucidate the function of the protein/gene of interest. The yeast two-hybrid (Y2H) method is a popular technique to identify protein-protein interactions in vivo1. In this system, two proteins (X and Y) whose interaction is to be tested are fused to the DNA-binding (DB) domain and transcriptional activation domain (AD), respectively. The DB-X fusion protein binds to a recognition sequence of the DB domain; therefore, when proteins X and Y interact, the AD-Y fusion protein comes into the proximity of the recognition sequence. Consequently, transcription of the reporter gene downstream of the recognition sequence is activated. Therefore, the presence or absence of reporter gene activity can be used to determine the presence or absence of the protein-protein interaction1.
Once a specific interaction partner of the protein of interest is identified, further analyses should be performed to elucidate the biological function of the interaction. For this purpose, if mutants of the proteins that impair or remove the specific protein-protein interaction can be isolated, they will serve as powerful tools. The Y2H system can be used directly to isolate such mutants by screening 'interaction-negative' clones, starting with the wild-type 'interaction-positive' clone. To accelerate this process, 'reverse' Y2H (rY2H) systems were developed2,3. In rY2H systems, the host yeast strains harbor counter-selectable marker genes as reporter genes, meaning yeast cells grow only when the AD-Y and DB-X proteins do not interact.
Although both the Y2H and rY2H systems allow the isolation of interaction-negative mutants, the process of isolating the mutants is laborious because not all of the candidates obtained by screening carry the desired type of mutations (usually missense mutations). The most serious issue is that a significant fraction of candidates harbor frameshift or nonsense mutations, and it is necessary to perform western blotting to exclude undesired clones. To overcome this problem, new plasmid vectors have been developed4. In these vectors, KanMX, a drug resistance marker, is positioned out-of-frame downstream of the DB domain or AD. The marker gene becomes in-frame with the DB domain or AD only when the gene of interest is inserted. When a random mutation(s) is introduced in the gene of interest, undesirable mutants, such as those with frameshift or nonsense mutations, can be easily eliminated by performing drug resistance selection, and candidates carrying desirable missense mutations can be easily identified with the Y2H screen4. This article presents a protocol to isolate interaction-null/impaired mutants of a protein of interest using this strategy.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M EDTA (8.0)</td>
			<td>Nacalai Tesque Inc.</td>
			<td>14347-21</td>
			<td></td>
		</tr>
		<tr>
			<td>10% SDS Solution</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>313-90275</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>135-07522</td>
			<td></td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Kishida Chemical Co. Ltd.</td>
			<td>110-64785</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-Gal)</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>021-07852</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Formedium</td>
			<td>AGR60</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin Sodium</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>68-52-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-HA-tag mAb-HRP-DirecT&#160;&#160;</td>
			<td>Medical &#38; Biological Laboratories Co. Ltd.</td>
			<td>M180-7</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA from salmon testes</td>
			<td>Merck KGaA.</td>
			<td>D1626</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck KGaA.</td>
			<td>9-0770-4-4L-J</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper for colony lift (Grade 50)</td>
			<td>Whatman, Cytiva</td>
			<td>1450-090</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper for colony lift (No.4A)</td>
			<td>Advantec Toyo Kaisha, Ltd.</td>
			<td>01411090</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper for replicaplating (No.1)</td>
			<td>Advantec Toyo Kaisha, Ltd.</td>
			<td>00011150</td>
			<td></td>
		</tr>
		<tr>
			<td>G-418 Sulfate</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>075-05962</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Kishida Chemical Co. Ltd.</td>
			<td>230-37585</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>163-03545</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Acetate Dihydrate</td>
			<td>Nacalai Tesque Inc.</td>
			<td>20604-22</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#8226;7H2O</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>131-00405</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#8226;12H2O</td>
			<td>Nacalai Tesque Inc.</td>
			<td>10039-32-4</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Nacalai Tesque Inc.</td>
			<td>31319-45</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4&#8226;2H2O</td>
			<td>Nacalai Tesque Inc.</td>
			<td>31717-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towel</td>
			<td>AS ONE Corp.</td>
			<td>7-6200-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:Isoamyl Alcohol 25:24:1</td>
			<td>Nacalai Tesque Inc.</td>
			<td>25970-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid DNAs</td>
			<td>the National BioResource Project - yeast (https://yeast.nig.ac.jp/yeast/top.xhtml)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid isolation Kit</td>
			<td>Nippon Genetics Co. Ltd.</td>
			<td>FG-90502</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Glycol #4,000</td>
			<td>Nacalai Tesque Inc.</td>
			<td>11574-15</td>
			<td></td>
		</tr>
		<tr>
			<td>SC double drop-out mix -Leu -Trp</td>
			<td>Formedium</td>
			<td>DSCK172</td>
			<td></td>
		</tr>
		<tr>
			<td>Seamless cloning kit (In-Fusion assembly )</td>
			<td>Takara Bio Inc.</td>
			<td>#639648</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>190-12865</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin Sulfate</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>3810-74-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq polymerase (GoTaq Green Master Mixes)</td>
			<td>Promega Corp.</td>
			<td>M7122</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS (hydroxymethyl) aminomethane</td>
			<td>Formedium</td>
			<td>TRIS01</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Nacalai Tesque Inc.</td>
			<td>12967-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>ThermoFisher scientific Inc.</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Nacalai Tesque Inc.</td>
			<td>35624-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>ThermoFisher scientific Inc.</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Nitrogen Base (YNB)</td>
			<td>Formedium</td>
			<td>CYN0210</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolyase 100T</td>
			<td>Nacalai Tesque Inc.</td>
			<td>07665-55</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating interaction-null&#47;impaired mutants using the yeast two-hybrid assay

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

Author Spotlight: Enhanced Isolation of Interaction-Null Mutants in Yeast

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

We are interested in understanding the regulatory mechanisms of the cell cycle in Eukaryotes, especially the relationship between DNA replication, stable germ maintenance, and cell proliferation. The progression of the cell cycle is regulated by cyclin-dependent kinases, or CDKs, in Eukaryotes. This is the case for the initiation of DNA replication.We have identified a way, on substrates of CDKs, to initiate DNA replication. To understand the function of the protein of your interest, a comparison of the wild-type and mutant is a direct and effective way. Yeast two-hybrid assay, being simple detection and will enhance the process of isolating interaction-null mutants.This will contribute to advancement of research in the field.

Recently, it was found that the C-terminal half of Pol2 protein (Pol2-C) interacts with Mcm10. Both proteins are essential for the initiation of DNA replication and, hence, for cell growth in the budding yeast Saccharomyces cerevisiae13,14,15. To help understand the biological significance of this interaction, Pol2-C mutants that have no/diminished interaction with Mcm10 were isolated using the method described here.</p...

Watch this Scientific Journal Video about Author Spotlight: Enhanced Isolation of Interaction-Null Mutants in Yeast at JoVE.com

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the ...

isolating-interaction-nullimpaired-mutants-using-yeast-two-hybrid

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265 Views.  Kochi University of Technology. We are interested in understanding the regulatory mechanisms of the cell cycle in Eukaryotes, especially the relationship between DNA replication, stable germ maintenance, and cell proliferation. The progression of the cell cycle is regulated by cyclin-dependent kinases, or CDKs, in Eukaryotes. This is the case for the initiation of DNA replication.We have identified a way, on substrates of CDKs, to initiate DNA replication. To understand the function of the protein of your interest, a...

Video: Author Spotlight: Enhanced Isolation of Interaction-Null Mutants in Yeast

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired...

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employment of two hybrid assay to identify interaction-impaired mutants in yeast

Employment of Two Hybrid Assay to Identify Interaction-Impaired Mutants in Yeast

generation of null mutants to elucidate the role of bacterial glycosyltransferases in bacterial motility

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial...

act1-cup1 assays determine the substrate-specific sensitivities of spliceosomal mutants in budding yeast

ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast

The ACT1-CUP1 assay, a copper growth assay, provides a quick readout of precursor messenger RNA (pre-mRNA) splicing and the impact mutant splicing factors have on spliceosomal function. This study provides a protocol and highlights the customization possible to address the splicing question of...

gene-targeted random mutagenesis to select heterochromatin-destabilizing proteasome mutants in fission yeast

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

This article describes a detailed methodology for random mutagenesis of a target gene in fission yeast. As an example, we target rpt4+, which encodes a subunit of the 19S proteasome, and screen for mutations that destabilize heterochromatin.

detection of cd40 protein-umbelliferone interaction via differential scanning fluorescence

Author Spotlight: Streamlining PCR Methods for Enhanced Accessibility and Efficiency

This protocol describes a unique method for detecting the binding between compounds and protein molecules, offering the advantages of minimal protein sample loss and high data...

High-Sensitivity Detection of Compound-Protein Binding Using Fluorescence

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high-throughput robotically assisted isolation of temperature-sensitive lethal mutants in chlamydomonas reinhardtii

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

Temperature-sensitive (ts) lethal mutants are valuable tools to identify and analyze essential functions. Here we describe methods to generate and classify ts lethal mutants in high throughput.

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

three-dimensional mapping of the rotation of interactive virtual objects with eye-tracking data

Author Spotlight: Insights into the Analysis of Human Interaction with 3D Virtual Objects

We have developed a simple, customizable, and efficient method for recording quantitative processual data from interactive spatial tasks and mapping these rotation data with eye-tracking...

Real-Time Recording and Mapping of Human Interaction with 3D Virtual Objects

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enhanced yeast one-hybrid screens to identify transcription factor binding to human dna sequences

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of &#62;1,000 TFs in a single...

isolation of umbilical cord-derived mesenchymal stem cells with high yields and low damage

Author Spotlight: Advancements in Stem Cell-Derived Vascularized Organoids and Optimized Isolation of Wharton&#039;s Jelly for Enhanced MSC Production

This study presents a unique blunt dissection procedure to preserve the integrity of Wharton's jelly (WJ), resulting in less damaged WJ and a greater quantity and viability of the harvested mesenchymal stem cells (MSCs). The method demonstrates superior yield and proliferative ability compared to conventional sharp dissection...

Dissection and Isolation of WJ-MSCs from the Human Umbilical Cord

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imaging of mthyper7, a ratiometric biosensor for mitochondrial peroxide, in living yeast cells

Author Spotlight: Advancing Mitochondrial Research - mtHyper7 Biosensor for Subcellular Analysis 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available...

Imaging of Mitochondrial Peroxide in Living Yeast Cells Using mtHyper7

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enhancing density maps by removing the majority of particles in single particle cryogenic electron microscopy final stacks

Author Spotlight: Optimizing Cryo-EM Analysis with CryoSieve for Enhanced Particle Selection Efficiency

An advanced particle selection method for cryo-EM, namely CryoSieve, improves density map resolution by removing a majority of particles in final stacks, as demonstrated through its application on a real-world...

Enhancing Cryo-EM Map Quality with CryoSieve Particle Selection

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a modified yeast-one hybrid system for heteromeric protein complex-dna interaction studies

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics...

microscopic varicocelectomy under local anesthesia as the treatment of varicocele

Author Spotlight: Advancing Varicocele Treatment Through Enhanced Surgical Techniques

Varicocele accounts for 35%-40% of infertility cases among men of reproductive age. The international standard treatment method is microscopic varicocelectomy, typically performed under general anesthesia. This study proposes and implements microscopic varicocelectomy under local anesthesia, which provides more humane care for patients and reduces the economic...

Microsurgical Varicocele Removal Under Local Anesthesia

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improved hysteroscopic resection of endometrial polyps using 6-fr micro-scissors and forceps

Author Spotlight: A Reproductive Hysteroscopy Approach for Complete Endometrial Polyp Removal and Enhanced Endometrial Receptivity

The novel technique presented here employs a combination of 6-Fr micro-scissors and forceps for hysteroscopic treatment of endometrial polyps, demonstrating encouraging outcomes for infertile patients afflicted with this...

Hysteroscopic Technique with Micro-Scissors and Forceps for Polyp Removal

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high-throughput optogenetics experiments in yeast using the automated platform lustro

Author Spotlight: Advancing Optogenetics Research Using Lustro 

This protocol outlines the steps for utilizing the automated platform Lustro to perform high-throughput characterization of optogenetic systems in...

Yeast Optogenetic Response Analysis Using the Lustro Automation Technique

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generation of marked and markerless mutants in model cyanobacterial species

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species

Introducing multiple genomic alterations into cyanobacteria is an essential tool in the development of strains for industrial and basic research purposes. We describe a system for generating unmarked mutants in the model cyanobacterial species Synechocystis sp. PCC6803 and marked mutants in Synechococcus sp.

acupuncture treatment in a mouse model of chronic hypoxia-induced cognitive dysfunction

Author Spotlight: Improving Anesthesia Protocols for Enhanced Mouse Acupuncture Research

Here, we describe a protocol for implementing mild anesthesia and acupuncture treatment on a chronic hypoxia mouse model and conducting behavioral tests to assess the cognitive alterations post-treatment.

Cognitive Improvement in Chronic Hypoxia Mice with Acupuncture

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visual and microscopic evaluation of streptomyces developmental mutants

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

Here we present protocols for novice researchers to initiate phenotyping for the pharmacologically important bacterial genus...

Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

To begin, obtain the host yeast strain, transformed with the bait plasmid. After introducing the mutant library into it, select a plate with 500 to 2000 colonies. Set two sheets of filter paper on a replica block and make several replicas of the plate, and incubate the plates at 30 degrees Celsius for one to two days.For the yeast two hybrid color assay, place a sheet of filter paper on the replica plate until it is completely moistened. Remove excess moisture using several sheets of paper towel, twice. Then, using tweezers, peel the filter paper off the plate from the edge and quickly immerse it in liquid nitrogen.Thaw the frozen filter paper on a dry paper towel with the colony side up. Once thawed, immediately transfer it onto another filter paper soaked with buffer. Cover the plastic tray containing the filter paper, put it into an airtight container, and incubate it at 37 degrees Celsius.Once blue color appears, place the filter paper on 500 microliters of stop solution taken in a plastic tray and immediately dry it on a fresh paper towel. The color assay produced blue or white dots, differentiating the mutants that did not interact with the bait.